Team:UPO-Sevilla/Notebook/09 22

From 2010.igem.org

September, 22nd

Assay Team

We analyzed the plates we spread the day before. In the half of them there was no colonies. It seemed that thin needles could not arrive the medium with bacteria.

We started another chemotaxis assay: testing the chemotaxis behavior of Pseudomonas putida KT2442. We used three chemotaxis chambers. Also in each cubicle there were two needles, one with chemoattractant and other with the same buffer that there was in the medium (control). Thin and thick needle were in different lines of the chambers to test the perfect conditions.

Chamber 1: Aspartate as attractant.

1mM

10mM

100mM

thin needle

A C

A C

A C

thick needle

A C

A C

A C

Chamber 2: Salicylate as attractant.

1mM

10mM

100mM

thin needle

S C

S C

S C

thick needle

S C

S C

S C

Chamber 3: We wanted to test if E. coli K12 expels aspartate in a medium with enough succinate, so we set up E. coli K12 inocula in minimal medium with succinate and in minimal medium with glucose. Then we picked up the supernatant of those inocula and used it like an attractant in needled. We used two controls: supernatant of glutamate grown E. coli K12 and minimal medium with succinate.

Overnatant of E. coli> K12 in MM + Succ

Overnatant of E. coli> K12 in MM + Glu

MM + Succ

thin needle

att C

att C

att C

thick needle

att C

att C

att C

Bactera of the medium in the chemotaxis chambers were at 0’07 of optic density (OD). We gave one hour to the bacteria to get into the needles. Then we spread 102 and 104 dilutions of the needles content in LB plates. It was grown overnight at 30ºC.